The resolution of protein mixtures containing ribonucleases may be accomplished using polyacrylamide gel electrophoresis. The catalylic property of RNases, expressed as polynucleotide hydrolysis, provides a sensitive measure of enzyme activity. The advantages of both these techniques are combined in polynucleotide-polyacrylamide gel electrophoresis in the following manner: 1) 7.5% polyacrylamide gels, crosslinked with N,N'-methylene-bis-acrylamide and containing 0.03-0.5% polynucleotide, are prepared. Any polynucleotide that does not precipitate as gelling occurs may be utilized. 2) Samples containing RNases are electrophoresed at a pH well above or below the pH optima of the RNases. Enzyme catalyzed hydrolysis of polynucleotide does not occur under these conditions. 3) After electrophoresis, incubation of the gel in buffer results in the degradation of polynucleotide in the narrow bands to which the RNases are confined. 4) Staining with pyronin Y yields a reddish-purple gel containing lightly colored or colorless regions indicating the presence of RNase activity. Incorporation of polyadenylic acid in the gels, as a substrate for electrophoresed normal human serum ribonucleases, led to the visualization of twelve bands of enzyme activity located between Rf 0.0 and 0.4. Two types of patterns, differing in the locations of enzyme activities within the gels, were found using normal serum (25 samples).